Anaplasma phagocytophilum is a tick-borne pathogen responsible for granulocytic anaplasmosis in humans (Bakken J. S., et al.: Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA 272: 212-218, 1994). There has been a steady rise in cases of anaplasma infections, alone or through co-infection with other tick-borne pathogens (Varde S., et al.: Prevalence of tick-borne pathogens in Ixodes scapularis in a rural New Jersey County. Emerg. Infect. Dis. 4: 97-99, 1998). Left unchecked, anaplasma infection can be a potentially fatal disease resulting from the targeting and replication of Anaplasma phagocytophilum within human neutrophils (Bakken J. S. et al.: JAMA 272: 212-218, 1994). Anaplasma phagocytophilum infection thus emerges as a significant healthcare concern.
Detection of anaplasma infection is crucial. Ideally, a diagnostic assay should be capable of detecting anaplasma infection at its earliest stages, when antibiotic treatment is most effective and beneficial. Traditional detection methods for anaplasma infection includes: (i) microscopic identification of morulae in granulocytes, (ii) PCR analysis using whole blood, (iii) isolation of the anaplasma bacterium from whole blood, and (iv) serological tests, particularly indirect immunofluorescence assay (IFA). Microscopic examination is tedious and prone to sampling error. PCR test is sensitive in detecting the tick-borne pathogen during the period of time when the pathogen is present in the blood of infected patients. IFA is most commonly used (Park, J., et al.: Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays. Clinical and Diagnostic Laboratory Immunology 10(6): 1059-1064, 2003), but this test often gives false positive results. Such results can be attributed in part to the use of whole-cell antigens because such proteins may be shared with other bacteria (Magnarelli, L. A., et al.: Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer. J. Wildlife Dis. 40(2): 249-258, 2004). When clinical symptoms are manifested or high and stable antibody titers to Anaplasma phagocytophilum are found in patient blood, it reaches a late infection stage and bypass the window of antibiotic treatment.
So far, there are only a few surface proteins on anaplasma pathogen that are used in diagnostic assay for immuno-responses (i.e., IgG and IgM responses). It is generally believed that outer membrane proteins in pathogens are target for eliciting an immuno-response because they may be the first to be exposed to immune cells of a host. Regarding the Anaplasma phagocytophilum species, U.S. Pat. No. 6,964,855 discloses the use of an outer membrane protein and its fragments in a detection assay. U.S. Pat. No. 7,304,139 discloses a major surface protein 5 (MSP5) and its use in a diagnostic test. The '139 patent discloses a few patient's reactivity towards MSP5 and it lacks any data relating sensitivity and specificity, let alone any IgG/IgM distinction. Zhi et al. discloses cloning and expression of an outer membrane protein of 44 kDa and its use in a Western immunoblot assay (J. Clinical Microbiology 36(6): 1666-1673, 1998). Both MSP5 and p44 are outer membrane proteins in Anaplasma phagocytophilum. To the best knowledge of the inventors, there is no report on using any intracellular protein as an antigenic protein, let alone it use in ELISA detection for Anaplasma phagocytophilum. 
In Agrobacterium tumefaciens, TIVSS consists of twelve (12) protein components. virB5 and a part of virB2 are proteins located on the outer surface of the pathogen. On the other hand, the rest of the TIVSS in Agrobacterium tumefaciens reside within the pathogen (See, FIG. 1). TIVSS in Agrobacterium tumefaciens may represent a prototype for TIVSS in other species. The number of TIVSS protein components varies among various different species in the family. TIVSS in Agrobacterium tumefaciens is believed to form a conduit for transportation of macromolecules (such as proteins) across the cell membrane. Anaplasma phagocytophilum is a phylogenetically distant species. TIVSS in Anaplasma phagocytophilum consists of eight (8) protein components. And the manner by which TIVSS proteins assembly and their respective functions in Anaplasma phagocytophilum is presently unknown. Flabio R. Araujo et al. recently reported that sera of cattle infected with Anaplasma marginale (a phylogenetically distant species of Anaplasma phagocytophilum) can recognize recombinant virB9, virB10, and elongation factor-Tu (EF-Tu). To the best of the inventor's knowledge, there is no information exists regarding the cloning and recombinant expression of the Anaplasma phagocytophilum TIVSS protein components.
There is a continuing need in the discovery of a novel antigen present in Anaplasma phagocytophilum that may be useful in sero-detection of this pathogen. The present invention cures all the above-mentioned defects and provides a useful detection assay for Anaplasma phagocytophilum infection. Disclosed herein are the cloning, expression, purification, and use of two recombinant type IV secretion system (TIVSS) proteins virB10 and virB11 (rTIVSS virB10 and rTIVSS virB11). Particular embodiments include the development of a diagnostic ELISA test useful for detecting IgM/IgG antibody responses to Anaplasma phagocytophilum. The present assay discriminates between Anaplasma phagocytophilum IFA-positive and IFA-negative patient samples with high sensitivity (>70%) and specificity (>90%) values.